N-glycosylation does not affect assembly and targeting of proglycinin in yeast.
Glycinin, a simple protein, and beta-conglycinin, a glycoprotein, are the dominant storage proteins of soybean and are suggested to be derived from a common ancestor. To investigate why glycinin does not require glycosylation for its maturation, we attempted N-glycosylation of proglycinin A1aB1b using site-directed mutagenesis and yeast expression system. An N-glycosylation consensus sequence Asn-X-Ser/Thr was created at positions 103, 183, 196, 284 and 457 in the variable regions being strongly hydrophilic revealed from the alignment of amino acid sequences of various glycinin-type proteins. Among five mutant proglycinins (Q103N, H183N, G198T, S284N, N459T), Q103N was fully glycosylated, and H183N and N459T were partly (around 20% of the expressed proteins), whereas others were barely or not glycosylated. The glycosylated proglycinin was susceptible to endo-beta-N-acetylglucosamidase and N-glycanase cleavages. N-glycosylation did not cause inconveniences to processing of signal peptide, assembly into trimers and targeting into the vacuoles. Thermal and trypsin sensitivity analyses of the glycosylated proglycinin suggested that N-linked glycan prevents protein-protein interaction but does not stabilize the protein conformation. The reason why glycinin does not require N-glycosylation for its maturation is discussed.
Katsube
T
T
Research Institute for Food Science, Kyoto University, Uji, Japan.
Kang
I J
IJ
Takenaka
Y
Y
Adachi
M
M
Maruyama
N
N
Morisaki
T
T
Utsumi
S
S
D052179:Antigens, Plant; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D005916:Globulins; D006023:Glycoproteins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011489:Protein Denaturation; D021382:Protein Sorting Signals / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000648:ultrastructure; D055314:Seed Storage Proteins; D030262:Soybean Proteins; D013025:Glycine max / Q000737:chemistry*; D014170:Transformation, Genetic / Q000235:genetics; D014357:Trypsin / Q000378:metabolism
D052179:Antigens, Plant; D005656:Fungal Proteins; D005916:Globulins; D006023:Glycoproteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; C020668:glycanase; D014357:Trypsin